CZ3000U1 - Pharmaceutical composition for immunomodulating and adjuvant treatment - Google Patents

Abstract

Pharmaceutical composition for immunomodulating and adjuvant therapy containing a metabolic stressor, 2-deoxy-D-ribose and further active components, i.e. ribose, DL-alanine, nicotonic acid, L-ascorbic acid, thiamin and glutamic acid amide is intended for immunomodulating and adjuvant therapy and for prevention of weakening of the immunity system.

Description

The invention relates to pharmaceutical immunomodulatory and adjuvant treatment and to the immune system.

means for preventing weakness

BACKGROUND OF THE INVENTION

The immunomodulatory agents currently used in the treatment of various immunodeficiencies, autoimmune and tumor diseases can be divided into several groups according to their composition and origin. An important group are, for example, thymic hormones obtained by extraction of animal tissues, which are distributed under the names Thym fraction V (manufactured by Hoffmann LaRoche), Tymodulin and Leucotropina (manufactured by Ellem), Tymunox (manufactured by Cilag), TFX (manufactured by Polfa). Another group of immunomodulatory agents that are given considerable attention, especially at the level of basic medical research, are cytokines, which are intrinsic regulators of the immune response at the cellular level. Genetic engineering technologies allow the production of recombinant proteins that are effective components of immunomodulatory agents such as e.g.

alpha2) manufactured by Hoffmann LaRoche, growth factor (G-CSF) of the same manufacturer marketed under the designation Neupogen. Today, classical interleukin-2 therapy has been discussed in detail at the IMUNOTHERAPY OF INFECTIONS congress (Berlin, 4.-7.5.1993), especially in terms of its failure in some patients. Leukocyte dialysates in the Czech Republic known as Transfer Factor (manufactured by SEVAC) are also popular immunomodulatory agents. Among the oldest and most frequently used are agents based on the adjuvant effect of selected antigens predominantly derived from membrane components

Roferon (interferon or hematopoietic derived from the components of bacteria membranes, eg Stava (SEVAC), Olimostimulin (Faculty of Medicine, University of Universty Palacky Olomouc). their effect at the level of modern European research (Immunotherapy of Infections, Berlin, May 4-7, 1993).

The composition known from the Greek Patent No. 72440 to Dr.T.H.Alivizatose, based on a mixture of D-ribose, DL-alanine, nicotinic acid and the balance of metabolism, organism, can be classified as an immunomodulatory effect.

The modern concept of ascorbic, according to the author restores which leads to the strengthening of immunity also among the means with significant immunomodulatory immunity of the organism, ie therapy based on defining the targeted interventions in the area and character of the effect of the pharmacological agent.

The elaboration of such a definition is very complicated and difficult for most immunomodulatory agents used hitherto. The causes of these difficulties are due to the fact that some immunomodulatory substances are obtained from biological sources (it is therefore difficult to define all of their possible biological activities), and because all of these substances exhibit a number of biological effects that prevent localization of the device system. Therapy based on the application of individual cytokines is, according to the current experience, associated with a number of risks arising from the involvement of autoregulatory mechanisms of the recipient of cytokines and consequently to the impaired performance of the immune system. It is also known that activation of the immune system by an exogenous cytokine is not fully feasible because of the lack of other natural foci. Thus, it would be highly desirable to provide an immunomodulatory agent activating the system at a physiological level, i.e. involving all natural control and regulatory circuits.

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The essence of the technical solution

The essence of the immunomodulatory solution is a pharmaceutical composition for and adjuvant treatment containing a metabolic stressor. The invention follows the Greek patent which extends, in the light of current knowledge, the regulation of the immune system by influencing it by metabolic stressors. Stressor is a natural, non-toxic compound, in this case a deoxy derivative of D-monosaccharides, which affects the production of cytokines, i.e., the regulators of the immune response at the cellular level.

The preparation is an isotonic solution of the following composition:

Responses and Metabolic

D-ribose 10-35 G 2-deoxy-D-ribose 10-35 G DL-alpha-alanine 2-12 G nicotinic acid Table 2-10 G L-ascorbic acid. 7-20 G thiamine 0.2-2 G glutamic acid amide 0.01- 15 g

dissolved in 1000 g of physiological saline.

The presence of deoxy derivatives of D-monosaccharides leads to targeted intervention in the immune system and allows the use of a pharmaceutical composition for the treatment of a broader spectrum of immune disorders and diseases, in particular autoimmune and tumor diseases than mentioned in the Greek patent cited.

Examples

Example 1

Quantitative composition of the pharmaceutical composition, Embodiment A:

g D-ribose g 2-deoxy-D-ribose g DL-alpha-alanine 2 g nicotinic acid 7 g L-ascorbic acid dissolved in 100 g of physiological saline.

Example 2

Effect of the composition of Embodiment A of Example 1 on the production of specific antibodies:

Specific antibodies were determined in the serum of C3H mice by ELISA on day 7 after ovalbumin immunization. Composition A was administered repeatedly at a dose of 5x0.2 ml i.p.

antibodies (mean-SD) % control control 672.8-55.62 100 ALIGN! test 1055.3-387.6 156.8

In this embodiment, the composition significantly increased the production of specific antibodies.

Example 3

Effect of the composition of Embodiment A of Example 1 on mitogen-induced splenocyte proliferation and mixed lymphocyte response:

The test was performed by the drop drop method, in the embodiment A was administered in a dose of 3x0.2 ml i.p.

Formulation to C3H mice.

The spleen was removed the day after the last administration and the test performed. Concanavalin A (CON A), lipopolysaccharide (LPS). IN

Phytohemagglutinin (PHA), a two-way mixed lymphocyte culture (MLR) splenocyte assay from DBA / 1 mice, was used as mitogens.

were used as aloantigen

dpm - SE % control CON A control 834.8-175.0 100 ALIGN! test 490.7- 70.9 58.8 PHA control 1065.4-306.0 100 ALIGN! test 593.7-125.9 55.7 LPS control 1068.9-274.4 100 ALIGN! test 521.8- 96.7 44.8 MLR control 494.2- 49.6 100 ALIGN! test 477.3- 97.2 96.6

Administration of the pharmaceutical composition in this embodiment did not stimulate CON A, PHA and LPS-induced lymphocyte proliferation, but inhibition of proliferation was observed.

3 g 5 g 35 g 12 g 10 g 20 g

Example 4

Quantitative composition of the pharmaceutical composition,

B:

D-ribose

2-deoxy-D-ribose DL-alpha-alanine L-ascorbic acid nicotinic acid dissolved in 1000 g saline.

Example 5

Effect of the pharmaceutical composition of Embodiment S on the production of specific antibodies:

Specific antibodies were determined in the serum of C3H mice by ELISA on day 7 after ovalbumin immunization. The composition of Embodiment B was administered repeatedly at a dose of 5x0.2 ml i.p.

antibodies (average-SD) % control control 672.3-55.62 100 ALIGN! test 592.4-95.4 88

The pharmaceutical composition of Embodiment B did not stimulate the production of specific antibodies.

Example 6

Effect of the pharmaceutical composition of Embodiment B on mitogen-induced splenocyte proliferation and mixed lymphocyte response:

The test was performed by the drop drop method. The composition was administered at a dose of 3x0.2 ml i.p. C3H mice. The spleen was removed the day after the last administration and a test was performed. Concanavalin A (CON A), Phytohemagglutinin (PHA) and lipopolysaccharide (LPS) were used as mitogen. In the two-way mixed lymphocyte reaction (MLR) assay, splenocytes from DBA / 1 mice were used as alloantigen.

dpm-SE % control CON A control 834.8-175.0 100 ALIGN! test 1109.4-225.3 132.9 PHA control 1065.4-306.0 100 ALIGN! test 1475.6-213.4 13 8,5 LPS control 1068.9-274.4 100 ALIGN! test 1500,6-258,2 140.4 MLR control 494.2- 49.6 100 ALIGN! test 411.0- 47.7 33.2

The pharmaceutical composition of Embodiment 3 did not significantly stimulate CON A, PHA, and LPS induced lymphocyte proliferation.

Note.

The so-called standard was not used in the tests because a suitable and generally valid comparative immunomodulatory agent has not been established by the World Health Organization (see paragraph Prior Art, the issue of defining the nature of an immunomodulatory device).

Example 7

Effect of the pharmaceutical formulation in Embodiment B on the growth of murine B16 melanoma:

C57B16 mice were transplanted subcutaneously with mouse B16 melanoma on the right flank. After engraftment, the pharmaceutical composition was applied to human / mouse body surface area according to Grossmann V., Jarkovska I., Kvetina J .: Pharmacy 9, 1983: 400-404, meaning that each mouse was always administered 0. 04 ml of the formulation in Embodiment B into the tail vein. The pharmaceutical composition was administered for 23 days a total of 20 times, with a two-day break after every fifth administration. The control group (10 mice transplanted with melanomen B16) was administered physiological saline in an equal volume instead of the pharmaceutical composition. Every three days, tumors were measured by caliper and the growth curve was followed. At the same time, the body weight of the mouse was also monitored. During treatment with the pharmaceutical composition of Embodiment B, a statistically significant difference in tumor sizes was found between the test and control groups. However, the difference between groups was not statistically significant at the end of therapy. In the following period, an optimal administration schedule will be sought and a correlation between the efficacy of the composition and the amount of tumor mass prior to initiation of therapy will be made.

Example 8

Effect of the pharmaceutical composition in Embodiment B on the course of rat leukemia:

In an orientation experiment, the progress of leukemia in SD / Ipcv rats was monitored by monitoring body weight, survival and blood counts. Haematological findings prior to initiation of administration of the pharmaceutical composition in Embodiment B:

 erythrocytes 8 050 000 leukocytes 12 300 platelets 826 000differential: blastic elements 4 myelocytes 0 metamyelocytes 6 rods of segments 25 eosinophils of monocytes ´ · 4 lymphocytes 56

X-cells 9

The preparation was applied to the tail vein in an amount corresponding to the body surface area (see Example 7).

After the 5th dose the following changes occurred in the haematological finding:

erythrocytes 7 350 leukocytes 10 platelets 982 differential: blastic elements myelocytes metamyelocytes rods segments eosinophils monocytes lymphocytes

X-cells

000

900

000

5 9

The haematological findings were monitored weekly, and the following values were found to be discontinued:

erythrocytes leukocytes thrombocvtv

310,000 9,300

162 000 differential: blastic elements 0 myelocytes o meta myelocytes 0 rods 2 segments 39 eosinophils 0 monocytes 6 lymphocytes 51

Following therapy, the follow-up was continued

weeks with the following haematological finding at the end of the reference period:

erythrocytes 8 700 000 leukocytes 8 400 platelets 778 000 differential:

400 blastic elements 0 myelocytes metamyelocytes rods segments eosinophils monocytes lymphocytes X-cells

Body weight, which did not undergo significant changes over 8 weeks, as well as the clinical condition of the test animal, was monitored throughout the experiment. It is apparent from the results presented that the hematological finding of the animal under investigation has been normalized as a result of therapy with the pharmaceutical composition of Embodiment B.

The present example currently serves as a starting material for a targeted study of the efficacy of a pharmaceutical composition on hematological cancer as well as osteoporosis under the conditions prescribed for pharmaceutical compositions intended for the treatment of cancer.

Example 9

Acute toxicity limit tests in mice and rats following a single i.v. and i.p. administration of the composition in Embodiment B:

NMRI mice at 6 weeks of age from conventional breeding were used for the experiment. Male body weight ranged from 31 to 37 g, females 27.5 to 32 g at i.v. administration and 32-37 g in males and 28-33.5 g in females for i.p. application.

The animals were treated with the formulation of Embodiment B at a dose of 2 ml /. 10Ό g for both applications. I.v. application was performed into the tail vein, i.p. into the lower left quadrant of the abdominal cavity. The animals were housed

in a climate controlled room with controlled light mode (12 hours light, 12 hours dark, room temperature 23-25 ° C, relative air humidity 50-70%), in polypropylene breeding cages (type T3 Velaz) in groups of 5 animals per female ,, males were housed individually in polypropylene breeding cages (type T2 Velaz) on a bed of sterilized shavings due to mutual aggression. Standard pellet diet ST 1 (Velaz) and drinking water were available ad libidum throughout the experiment.

The clinical health and behavior of the animals were monitored daily for 21 days after administration, and the body weight of the animals was monitored once a week. At the end of the study period, all animals were subjected to a macroscopic pathological examination.

The study was conducted according to the internationally applicable OECD regulations for acute toxicological studies.

Clinical observations: i.v. intensive regular breathing was observed immediately after application, up to 30 minutes after application, as well as animals with no signs of symptoms throughout the study period. Also, the growth curves showed no evidence of a possible toxic effect of the pharmaceutical composition. When i.p. deeper breathing, spastic walking with a hint of write convulsions were observed after application. Within 30 minutes, as in the entire observation period, the animals were free of apparent clinical signs. There was no death of mice in either group during the experiment, both at the application

at the end of the 21-day period, the animals were subjected to a macroscopic pathological examination. When i.v. There were no macroscopic deviations from the standard on the abdominal and thoracic organs in both the male and female application, and the application site was also free of pathological findings. Findings in i.p. The method of administration was identical for both sexes, a whitish coating on the surface of the spleen, partly or completely adhering to the stomach, the kidneys lighter.

According to the above results, a limit test of acute clinical essential monitored

 Toxicity performed according to OECD international guidelines for acute toxicological studies of the pharmaceutical formulation at a dose of 20 ml / kg at i.v. and i.p. administration in mice of both sexes showed no toxic symptoms and was also free from death.

To the next. Wistar rats at 6 weeks of age from conventional breeding were used in the experiment. The body weight of male rats ranged from 194 to 209 g, females ranged from 159 to 170 g at i.v. application and 190 to 215g in males and 161 to 175g in females for i.p ..

The animals were administered the composition of Embodiment B at a dose of 2 ml / 100g for both routes of administration. I.v. application was carried out into the tail vein, i.p. application to the upper left quadrant of the abdominal cavity. The animals were housed in a climate controlled room (12 hours light, 12 hours dark, room temperature 23-25 ° C, relative humidity 50-70%), in polypropylene breeding cages (type T4 Velaz) in groups of 5 same-sex animals on a bed of sterilized shavings. Standard pellet diet ST 1 (Velaz) and drinking water were available ad libidum at all times.

The clinical health and behavior of the animals were monitored daily for 21 days after administration, and the body weight of the animals was monitored once a week. At the end of the study period, all animals were subjected to a macroscopic pathological examination.

The study was conducted according to the internationally applicable OECD regulations for acute toxicological studies.

Clinical observations: i.v. i i.p. intensive regular breathing and mild slackness were observed immediately after administration, which persisted within 5 hours after administration. During the remainder of the study period, the animals were free of any apparent clinical signs. Also, the growth curves did not show any significant changes suggesting the possible toxic effect of the pharmaceutical composition of interest. In the course of the experiment, no animal was killed in both routes of administration.

At the end of the 21-day observation period, the animals were subjected to a macroscopic pathological examination. When i.v. application were not found in males, as well as females, macroscopic deviations from the norm on the organs of the thoracic cavity, also organs? abdominal cavity except for recurrent findings of lighter kidneys and the site of application was free of pathology. Findings in i.p. The method of application was the same for both males and females: on the surface of the spleen an off-white coating, this partially or completely adhered to the stomach, the lobes of the liver usually coalesced, also with whitish coatings, lighter kidneys.

Histological examinations performed beyond the scope of OECD regulations identified liver findings as focal chronic productive perihepatitis, with liver parenchyma without pathological findings. The findings on the liver were within the norm, in some cortical epitheliums a hyaline deposit was found.

According to the above results, an acute toxicity limit test conducted according to OECD guidelines for acute toxicological studies of a pharmaceutical composition of Embodiment B at a dose of 20 ml / kg at i.v. and i.p. administration in rats of both sexes did not produce any significant toxic symptoms and was also free from death.

Claims (4)

A pharmaceutical composition for immunomodulatory and adjuvant therapy, comprising: D-ribose 10 - 35 G 2-deoxy-D-ribose i 10 - 35 G DL-alpha-alanine 2-12 G nicotinic acid Table 2-10 G L-ascorbic acid 7-20 G thiamine 0.2 - 2 G glutamic acid amide 0.01- 15 G dissolved in 1000 g of physiological saline. A pharmaceutical composition according to claim 1, i.e., in a form suitable for i.v. application. The pharmaceutical composition of claim 1, having a form suitable for oral use. · Use of an active ingredient, i.e. 2-deoxy-D-ribose, for the manufacture of a pharmaceutical composition according to claims 1-4.